1. Field of the Invention
The present invention relates generally to the fields of diagnosis and treatment to prevent the onset of AIDS. More particularly, it concerns the use HIV peptides and HLA-restricted T-cell responses in both prediction of long-term non-progression of AIDS and prevention of AIDS.
2. Description of Related Art
During progressive human immunodeficiency virus type 1 (HIV-1) infection, the virus-specific immune responses of an infected subject gradually deteriorate, leading to the development of acquired immunodeficiency syndrome (AIDS). Most infected patients do not exhibit overt clinical manifestations of the disease for six to ten years following initial infection. Reports indicate, however, that approximately 5% of HIV-1 infected persons remain free of disease for ten or more years. (Haynes, 1996; Munoz, 1995; Rinaldo, 1995; Rowland-Jones, 1995; Rowland-Jones, 1993; Clerici, 1991; Lifson, 1991). Such a person, termed a long-term non-progressor (LTNP), exhibits lower viral loads and stable CD4+ cell counts.
The induction of a cytotoxic T-lymphocyte (CTL) response constitutes a significant defense mechanism against viral infections; occasionally, a virus-specific CTL response can render full protection without a concomitant antibody response (Sastry 1992; Bevan, 1989; Lukacher, 1984). Recent studies suggest the importance of cell-mediated immunity (CMI) for maintenance of disease-free status in an LTNP and in individuals belonging to high-risk groups (Rosenberg, 1997; Haynes, 1996; Munoz, 1995; Rinaldo, 1995; Roos, 1995; Rowland-Jones, 1995; Koup, 1994; Pantaleo, 1994; Rowland-Jones, 1993; Picard, 1992; Clerici, 1991; Lifson, 1991). Importantly, a small number of apparently uninfected children born to HIV-infected mothers and HIV-exposed but uninfected Gambian women have demonstrated HIV-specific cytotoxic T lymphocyte (CTL) responses (Rowland-Jones, 1995; Rowland-Jones, 1993). Also, Rinaldo et al reported that both high levels of anti-HIV-1 memory CTL activity and low viral loads are associated with the lack of disease in HIV-1-infected LTNPs.
The immune system may effectively eliminate virus-infected cells during the clinical course of HIV-1 infection using virus-specific major histocompatibility complex (MHC) class-I restricted CTL activity (Koup, 1994). The above evidence suggests that HIV-1-specific CTL activity is important for controlling viral spread during the clinical course of HIV-1 infection (Klein, 1995; Koup, 1994), for maintaining low levels of viral load during the asymptomatic phase (Musey, 1997; Rinaldo, 1995; Koup, 1994; Walker, 1987), and possibly for complete elimination of virus-infected cells, as implied from the observation of HIV-exposed, but virus-negative, children and women (Rowland-Jones, 1995; Rowland-Jones, 1993). Furthermore, observations from cross-sectional studies have shown the absence, or severely decreased levels, of HIV-1-specific CTL responses during advanced stages of HIV-1 infection (Carmichael, 1993). Therefore, researchers have focused on identifying virus-specific CTL epitopes.
The induction of specific CTL responses in the context of human MHC class I antigens has been demonstrated by many investigators with respect to HLA-A and HLA-B. HLA-A and -B act as strong transplantation antigens and as restriction molecules for recognition of foreign antigens by CTLs (Dill, 1988; McMichael, 1977). In contrast, little is known about the functional properties of the third class I antigen, HLA-C. HLA-C antigens are encoded by a DNA sequence that is closely related to the sequences encoding HLA-A and -B and lies between them. HLA-C antigens are expressed on lymphoid cells, although to a lesser extent (approximately 10%) than either HLA-A or -B (Schendel, 1992; Gasson, 1987; Sodoyer, 1984).
Recent reports suggest that expression of HLA-C confers protection against lysis by natural killer (NK) cells and also by non-MHC-restricted effector T cells (Falk, 1995; Falk, 1993). In particular, expression of Cw7 was demonstrated to govern directly resistance to lysis against both these types of effector populations (Falk, 1995).
Typically, induction of virus-specific CTLs can be effected by infection with a virus or recombinant virus that expresses a viral gene product. The viral gene product is processed and presented as a peptide on the surface of infected cells in association with an MHC class I molecule for recognition by the CTL (Unanue, 1989; Branciale, 1987).
Additionally, research efforts have concentrated on identifying and characterizing HIV peptides that elicit a viral-specific CTL response. Townsend et al. illustrated the concept of using T-cell epitopes in proteins as vaccine candidates when their group demonstrated the use of short synthetic peptides from influenza nucleoprotein as epitopes for CTL responses. The inventors and others have reported using synthetic peptides to generate virus-specific CTLs in vivo (Kast, 1991; Aichele, 1990; Deres, 1989; Sastry, 1992; Sastry, 1994; Casement, 1995) against influenza, lymphocytic choriomenengitis, Sendai virus and HIV. HIV-infected patients or humans and mice immunized with HIV proteins exhibit a specific CTL response against various HIV gene products (Chenciner, 1989; Tsubota, 1989; Nixon, 1988; Walker, 1988; Plata, 1987; Walker, 1987).
The identification and characterization of additional HIV-specific HLA haplotypes and HIV peptides capable of inducing a specific CTL response would be useful for the diagnosis and treatment of AIDS, particularly if the haplotypes were related to the disease-free status of LTNPs and to peptides from highly conserved HIV sequences.
The invention generally relates to diagnostic, preventative, and treatment therapies of AIDS. The present invention provides a method of predicting long-term non-progression in an HIV-infected patient. The invention also provides a method of preventing AIDS in both infected and uninfected subjects. It is based on the observation that an HLA-C-specific CTL response can be demonstrated against some HIV envelope peptides.
The present invention first provides a method for predicting long-term non-progression in an HIV-infected patient by determining whether the patient demonstrates an HLA-Cw7 CTL response against a target cell. In one embodiment, the patient is infected with, or at risk of infection by, HIV-1. Methods of assaying for the existence of an HLA-Cw7-restricted CTL response comprise obtaining cells from a patient and exposing them to target cells that express the HLA-Cw7 haplotype. The invention is understood to include cells obtained from peripheral blood mononuclear cells (PMBC), mucosal lymphocytes, lymph node cells, and spleen cells. In another embodiment, the PMBCs are stimulated with phytohemagglutinin, anti-CD3 antibody, or HIV antigens prior to exposing them to target cells.
An HIV-infected subject may be tested for an HLA-Cw7-restricted CTL response or possession of the HLA-Cw7 haplotype. The CTL response also can include CD4- and CD8-expressing (CD4+ and CD8+) cells. The method for detecting an HLA-Cw7 restricted CTL response uses target cells that include cells from an autologous B cell line, dendritic cells, or MHC-matched cells.
The CTL response can be assayed by lysis of the target cell, which could be labeled using [51Cr]sodium chromate, or by production of xcex3-interferon, or by tetramer assay.
In another embodiment, the method of the present invention provides a target cell that presents at least an HIV polypeptide, which includes the HIV envelope (env) polypeptide or the gag polypeptide, in addition to HIV polypeptide fragments thereof. In a preferred embodiment, the polypeptide is gp160, or fragments thereof. In further embodiments, the invention predicts long-term non-progression of AIDS by using a target cell that presents a synthetic peptide whose amino acid sequence is derived from an HIV gene product such as a synthetic peptide, which can be from 11 to 25 residues in length. In additional embodiments, the peptide sequences include YL(R/K)DQQLLGIWGC (SEQ ID NO:33 or SEQ ID NO:34), FLGFLGAAGSTMGAASLTLTVQARQ (SEQ ID NO:20), or VYYGVPVWKEA (SEQ ID NO:1).
Furthermore, the present invention includes the delivery of HIV peptides to the target cell by an expression construct that comprises a polynucleotide sequence encoding at least one HIV peptide under the transcriptional control of a promoter. In some embodiments, the expression vector is a viral vector. Such a viral vector can be from any virus selected from a group consisting of vaccinia virus, adenovirus, herpesvirus, retrovirus, adeno-associated virus and lentivirus.
The present invention next provides a method of preventing an HIV-infected subject from developing AIDS by determining whether the patient expresses HLA-Cw7 and demonstrates an HLA-Cw7-restricted, HIV-specific CTL response; if such a response is exhibited, the patient is administered a composition that contains an HIV polypeptide that is also an HIV CTL epitope. Alternatively, the methods of the invention can be practiced by determining whether the HIV-infected subject has an HLA-Cw7 haplotype. The method is understood to encompass patients who are infected with HIV-1. A composition of the claimed invention includes HIV polypeptides such as the env polypeptide, the gag polypeptide, and fragments of either. Furthermore, the HIV polypeptide of the claimed invention further is understood to include a synthetic peptide whose sequence is derived from HIV gene products. Such a synthetic peptide can be from 11 to 25 residues in length and could include sequences such as YL(R/K)DQQLLGIWGC (SEQ ID NO:33 or SEQ ID NO:34), FLGFLGAAGSTMGAASLTLTVQARQ (SEQ ID NO:20), or VYYGVPVWKEA (SEQ ID NO:1). The method also could include administering a plurality of HIV polypeptides. This plurality of HIV peptides could include 2 or more different peptides containing the sequences YL(R/K)DQQLLGIWGC (SEQ ID NO:33 or SEQ ID NO:34), FLGFLGAAGSTMGAASLTLTVQARQ (SEQ ID NO:20), or VYYGVPVWKEA (SEQ ID NO:1). Alternatively, the method could include administering one or more synthetic peptides from 11 to 25 residues in length that include sequences such as YL(R/K)DQQLLGIWGC (SEQ ID NO:33 or SEQ ID NO:34), FLGFLGAAGSTMGAASLTLTVQARQ (SEQ ID NO:20), VYYGVPVWKEA (SEQ ID NO:1), LWDQSLKPCVKLT (SEQ ID NO:4), SVITQACSKVSFE (SEQ ID NO:8), or GTGPCTNVSTVQC (SEQ ID NO:16). A plurality of peptides that comprises, two, three, four, five or all six of these sequences is included within the methods of the present invention. It is further contemplated that the sequences may also be included in peptides that have additional residues flanking one or more ends of the sequences. For example, the peptide FLGFLGAAGSTMGAASLTLTVQARC (SEQ ID NO:35) falls within the scope of the present invention.
The composition containing an HIV polypeptide may be administered with the HIV polypeptide coupled to a carrier molecule such as KLH or BSA. The composition could also include an adjuvant where the adjuvant is a lipid, a toxin, a cytokine, oligonucleotides or bacterial DNA.
A method of preventing an HIV-infected subject from developing AIDS also includes administering to the HIV-infected subject AZT or treating the HIV-infected subject with highly active retroviral therapy (HAART).
The present invention also provides a method for preventing an HIV-infected subject from developing AIDS when the subject does not exhibit an HLA-Cw7-restricted CTL response. In such a situation, the method includes first determining whether the subject has or expresses the HLA-Cw7 haplotype. Such a determination is understood to include conducting a serological assay using an antibody that recognizes HLA-Cw7 or performing a nucleic acid amplification reaction whereby an HLA-Cw7 region is amplified. If these tests reveal that the subject does express the HLA-Cw7 haplotype, a method of the claimed invention further provides that an HLA-Cw7 restricted CTL response be elicited. Ways of eliciting such a T-cell response include administering to the subject a therapeutically effective amount an interferon, particular xcex1- or xcex3-interferon, so that expression levels of HLA-Cw7 haplotype increase. This method also comprises the additional step of stimulating HIV-specific T helper cell responses.
The present invention also includes a method of preventing HIV infections in an uninfected subject by first determining whether the subject has or expresses an HLA-Cw7-hapiotype and, if the subject does, then administering to the subject a composition containing an HIV polypeptide that also is a CTL epitope, optionally also providing a T helper epitope. This preventative method contemplates prevention of infection by HIV-1. If the subject who is uninfected can express an HLA-Cw7 haplotype, the invention is understood to include compositions of an HIV polypeptide encompassing HIV envelope polypeptide or gag polypeptide, or fragments thereof. A synthetic peptide whose sequence is derived from an HIV polypeptide also can be used. This HIV-derived synthetic peptide can be from 11 to 25 residues in length and include the sequence YL(R/K)DQQLLGIWGC (SEQ ID NO:33 or SEQ ID NO:34), FLGFLGAAGSTMGAASLTLTVQARQ (SEQ ID NO:20), or VYYGVPVWKEA (SEQ ID NO:1). As previously mentioned, the methods of the present invention also include peptides comprising one or more of the following sequences: LWDQSLKPCVKLT (SEQ ID NO:4), SVITQACSKVSFE (SEQ ID NO:8), or GTGPCTNVSTVQC (SEQ ID NO:16). Any combination of one, two, three, four, five, or six of these peptide sequences may be used with the methods of the present invention. Furthermore, the HIV polypeptide can be coupled to a carrier such as KLH or BSA; and, it could also be administered with an adjuvant, where the adjuvant is a lipid, a toxin, cytokine synthetic oligonucleotide or bacterial DNA. In addition to administering to the uninfected subject an HIV polypeptide, the subject also can be treated with AZT or HAART.
As previously mentioned, the HIV peptides used in the methods of the present invention may be provided to a cell as an expression construct that comprises a polynucleotide encoding one or more HIV peptides. In some aspects of the present invention, different mini-gene constructs may be administered such that more than one type of peptide sequence is provided to a cell. In other aspects of the present invention, an expression construct may contain sequences that enable it to express more than one peptide sequence; for example, the expression construct may contain sequences that allow it to express both FLGFLGAAGSTMGAASLTLTVQARQ (SEQ ID NO:20) and VYYGVPVWKEA (SEQ ID NO:1). The expression construct may thus be able to express one, two, three, four, five, six or more peptide sequences.
In addition, the present invention includes preventing HIV infection in an uninfected subject when the subject expresses or can express the HLA-Cw7 haplotype and by eliciting an HLA-Cw7-restricted CTL response. Alternatively, the subject could be given a therapeutically effective amount an interferon so that the expression levels of HLA-Cw7 haplotype increase.
The use of the word xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d when used in conjunction with the term xe2x80x9ccomprisingxe2x80x9d in the claims and/or the specification may mean xe2x80x9cone,xe2x80x9d but it is also consistent with the meaning of xe2x80x9cone or more,xe2x80x9d xe2x80x9cat least one,xe2x80x9d and xe2x80x9cone or more than one.xe2x80x9d
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.